The cornea is the most anterior portion of the eye and creates the interface between the eye and the external environment. Due to its location, the cornea acts as a barrier, shielding the inner eye from foreign debris. The cornea must also allow light to pass through to the lens and, ultimately, the retina. The cornea is responsible for a majority of the light refraction into the eye.
The cornea is a highly ordered tissue. FIG. 12 depicts the layers of the cornea: the epithelium, Bowman's layer, the stroma, Descemet's membrane, and the endothelium, from anterior to posterior. The epithelium is made up of six to eight layers of epithelial cells. The newest, youngest, cells are the most posterior and move up through the layers to the most anterior cornea. This makes the cells at the anterior cornea the oldest, and these are swept away in the tear film as they age (Oyster and Clyde, “The Cornea and the Sclera,” in The Human Eye: Structure and Function. Sinauer Associates, Inc.: Sunderland, Mass.; 1999. Pages 325-371). At its thickest portion, the epithelium accounts for 10% of the total corneal thickness. The epithelium acts as a water barrier between the external environment (including tears) from the stroma (Id). The cornea is avascular, but the epithelium contains a large number of nerve endings that respond to pressure stimuli.
The cornea is repaired by limbal cells, which are epithelial progenitor cells (Id.). Limbal cells are located in the conjunctiva, as shown in FIG. 13. The cells migrate laterally from the conjunctiva to the cornea, where they differentiate into epithelial cells.
Bowman's layer connects the epithelium to the stroma. The stroma is made up of ordered layers of type I collagen that account for roughly 90% of the cornea (Id). Fibroblast cells, also known as keratocytes, inhabit and organize the layers of collagen. The stroma provides the structural and mechanical support that the cornea requires. It also helps maintain the proper shape for the refraction of light into the lens.
Binding the endothelium to the stroma is Descemet's membrane, a protein layer that acts as a basement membrane for the endothelium. It is roughly three to four micrometers thick, about the same as the endothelium (Id.). The endothelium is a very metabolically active layer between the aqueous and the stroma. It acts mainly as a water pump to keep the stroma dry, which maintains the clarity of the cornea (Id.).
The three cell types of the cornea (epithelial, fibroblast, and endothelial) can be isolated using techniques described by Orwin and Hubel (Orwin et al., 2000 Tissue Eng. 6:301-19). This technique involves mechanical removal of the endothelial cells, and removal of the fibroblast and epithelium using the proteins collagenase and dispase, respectively. Limbal cells can also be extracted using the knowledge that they readily migrate from the conjunctiva.
Millions of patients worldwide are afflicted by corneal blindness. Failure of the cornea can result from various pathologies (keratoconus, Fuchs' dystrophy, post-cataract surgery complications, trauma, infection, etc.), and contributes significantly to morbidity and disability. Additionally, in the battlefield, combat injuries to the head and neck outnumber torso injuries by nearly 4 to 1. The eyes (and surrounding orbit) are the most frequently injured regions of the soldier's head and neck (Miyashita et al., 2006 J. Biomed. Mater. Res. B Appl. Biomater 76:56-63; Doillon et al., 2003 Int. J. Artif. Organs 26:764-73; Duan and Sheardown, 2006 Biomaterials 27:4608-17; Bryans et al., 2000 J. Sol-Gel SciTech 17:211-217). These injuries are significant sources of morbidity and mortality in fighting troops.
The cornea is a complex and unique tissue that is avascular and is predominantly composed of extremely ordered collagen lamellae. The purpose of the cornea is to refract light and transmit it to the retina. For optimal function, the cornea must be transparent (Freund et al., 1986 J. Optical Soc. Am. 3:1970-1982). In addition, the cornea is a connective tissue that must withstand intraocular pressure, the normal range of which is 10-21 mm Hg (Pinsky and Datye, 1991 J. Biomechanics 24:907-922), and constantly changing environmental conditions (ambient temperature, pH of the tear fluid, ultraviolet (UV) exposure, exposure to chemical fumes, dust, etc.). The biomechanical characteristics of the cornea are also very important.
Corneal transplants are commonly used to treat corneal disease and injury. Indeed, the cornea is the most commonly transplanted tissue in the United States with over 46,000 transplants performed annually (EBAA, “EBAA releases 2004 statistical report on eye banking. Washington DS: Eye Bank of America,” 2005:1-2). The worldwide demand for corneal tissue suitable for transplantation, however, cannot be met by the supply (generally, a total of 120,000 transplants are available for 10,000,000 patients, as reported by the World Health Organization (Consultation Meeting on Transplantation with National Health Authorities in the Western Pacific Region: World Health Organization, 2005)), and a similar situation may present itself in United States in the next few years (Eglin et al., 2005 Biomed. Mater. Eng. 15:43-50; Freund et al., 1986 J. Optical Soc. Am. 3:1970-1982; Pinsky and Datye, 1991 J. Biomechanics 24:907-922; Buzard, 1992 Refract. Corneal Surg. 8:127-38; Shah et al., 2008 Pediatric Research 63:535-544).
In order to address the pressing need for corneal implants, medical researchers have turned to corneal substitutes. However, the combination of strength and transparency of the cornea is unique, and represents a significant challenge when recreating this tissue in vitro. Synthetic corneal substitutes (keratoprostheses) are used to a limited extent in clinical practice. Current indications for keratoprostheses include corneal scarring from severe ocular surface disorders, and multiple failed corneal transplants. However, artificial corneas in current use, including those made of either polymethylmethacrylate (PMMA) or poly(2-hydroxyethylmethacrylate) (pHEMA), do not bio-integrate with surrounding tissues, nor do they allow epithelialization of their surface. Keratoprostheses made of pHEMA have a porous skirt to promote bio-integration, but neither true biointegration nor epithelialization of the prosthesis surface occurs. Consequently, complications such as host tissue melting, extrusion, and intraocular infection are common (Eglin et al., 2006 J. Mat. Chem. 16:2220-2230; Heinmann et al., 2007 Adv. Engr. Mats. 9:1061-1068). There is, therefore, tremendous interest for developing a tissue-engineered cornea (Lang et al., 1981 Current Eye Research 1:161-7; Liu et al., 2006 Invest. Ophthalmol. Vis. Sci. 47:1869-1875; Kelley et al., 1984 Invest. Ophthalmol. Vis. Sci. 25:1061-4; Balm et al., 1982 Ophthalmology 89:687-99; Evans et al., 2002 Biomaterials 23:1359-67; Ma and Bazan, 2000 Invest. Ophthalmol. Vis. Sci. 41:1696-702).